A proposed design for the cryopreservation of intact and adherent human embryonic stem cell colonies

Summary Recently, it was demonstrated that the application of slow-cooling cryopreservation protocols to adherent human embryonic stem (hES) cell colonies, cultured on matrigel or murine embryonic fibroblast feeder layers, resulted in marked improvement in postthaw viability and reduction in cell differentiation. However, the use of commercially available culture plates for this purpose presents several limitations. Most obviously, these plates are not designed for cryopreservation or to withstand the low temperatures encountered during liquid nitrogen cryopreservation, or both. The physical storage of cryopreserved plates is another consideration, in addition to difficulty in maintaining sterile conditions in liquid nitrogen storage and during the thaw phase in a water bath. Hence, a redesign of the cell culture plate for the cryopreservation of adherent hES cell colonies is proposed. In this model, a culture plate made of synthetic materials resistant to storage at −196° C of liquid nitrogen is designed, with readily attachable screw-cap culture wells that function as a replacement for cryovial storage. The detachable wells facilitate storage and after thawing can easily be reattached to a specially designed holding plate. Currently, there are no commercially available cell culture plates using this design concept. The proposed design is envisioned to facilitate the cryopreservation of intact adherent hES cell colonies that could assist the development of automated systems for handling bulk quantities of cells.     

High-throughput SNP genotyping by single-tube PCR with Tm-shift primers

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.     

Importance of arginine 20 of the swine vesicular disease virus 2A protease for activity and virulence

A major virulence determinant of swine vesicular disease virus (SVDV), an Enterovirus that causes an acute vesicular disease, has been mapped to residue 20 of the 2A protease. The SVDV 2A protease cleaves the 1D-2A junction in the viral polyprotein, induces cleavage of translation initiation factor eIF4GI, and stimulates the activity of enterovirus internal ribosome entry sites (IRESs). The 2A protease from an attenuated strain of SVDV (Ile at residue 20) is significantly defective at inducing cleavage of eIF4GI and the activation of IRES-dependent translation compared to the 2A protease from a pathogenic strain (J1/73, Arg at residue 20), but the two proteases have similar 1D-2A cleavage activities (Y. Sakoda, N. Ross-Smith, T. Inoue, and G. J. Belsham, J. Virol. 75:10643-10650, 2001). Residue 20 has now been modified to every possible amino acid, and the activities of each mutant 2A protease has been analyzed. Selected mutants were reconstructed into full-length SVDV cDNA, and viruses were rescued. The rate of virus growth in cultured swine kidney cells reflected the efficiency of 2A protease activity. In experimentally infected pigs, all four of the mutant viruses tested displayed much-reduced virulence compared to the J1/73 virus but a significant, albeit reduced, level of viral replication and excretion was detected. Direct sequencing of cDNA derived from samples taken early and late in infection indicated that a gradual selection-reversion to a more efficient protease occurred. The data indicated that extensive sequence change and selection may introduce a severe bottleneck in virus replication, leading to a decreased viral load and reduced or no clinical disease.     

Physical Activity Energy Expenditure of Adolescents in India

Physical activity (PA) has rarely been quantified in adolescent populations undergoing economic transition; therefore relationships with disease still remain uncertain. This study assessed whether absolute PA energy expenditure (PAEE), PAEE/kg, and PAEE/kg_FFM could be accurately estimated using accelerometry and a questionnaire in Indian adolescents and how these values compared to those of other populations. PAEE was assessed using doubly labeled water (DLW) in 30 adolescents from Chennai, India, over seven consecutive days, simultaneous with the measurement of PA using accelerometry and a previous‐week recall questionnaire. Accelerometry counts (regression analysis) and questionnaire data were used to estimate PAEE; estimates were cross‐validated using the Bland‐Altman method. Accelerometry data and DLW‐derived PAEE were visually compared to values from four North American and European populations. For boys, 49% of the variance in DLW‐derived PAEE was explained with an equation including accelerometry counts and fat‐free mass (FFM). Questionnaire‐derived estimates did not contribute to the explained variance in DLW derived PAEE. The group‐level PA of these Indian adolescents was successfully assessed using accelerometry, but not questionnaire. DLW‐derived PAEE/kg_FFM (mean (s.d.): 53.0 (27.5) kJ/kg_FFM/day) was lower in this group than other adolescent populations in Europe and similar to those in North America. Additionally, four boys and none of the girls accumulated ≥60 min/day of accelerometry‐derived moderate intensity activity, indicating low levels of PAEE and PA in these adolescents. Further research is necessary to investigate the association between PA and health outcomes in Indian adolescents.     

The Impact of Health Behaviours on Incident Cardiovascular Disease in Europeans and South Asians – A Prospective Analysis in the UK SABRE Study

Background

There is consistent evidence on the impact of health behaviours on risk of cardiovascular disease (CVD) in European populations. As South Asians in the UK have an excess risk of CVD and coronary heart disease (CHD) compared to Europeans, we investigated whether a similar association between combined health behaviours and risk of CVD and CHD among this high-risk group exists, and estimated the population impact.

Methods and Findings

In a prospective cohort of 1090 Europeans and 1006 South Asians (40–69 y) without prevalent CVD at baseline (1988–1990), followed up for 21 years to 2011, there were 601 incident CVD events [Europeans n = 255; South Asians n = 346] of which 520 were CHD events [n = 207 and 313 respectively]. Participants scored between 0 to 4 points for a composite score including four baseline healthy behaviours (non-smoker, moderate alcohol intake, physically active, frequent fruit/vegetable intake). Adjusted hazard ratios (95% confidence intervals) for incident CHD in Europeans who had three, two, one, and zero compared to four health behaviours were 1.33 (0.78–2.29), 1.96 (1.15–3.33), 1.36 (0.74–2.48) and 2.45 (1.18–5.10), respectively, p-trend = 0.025. In South Asians, corresponding HRs were 2.88 (1.33–6.24), 2.28 (1.06–4.91), 3.36 (1.53–7.39) and 3.48 (1.38–8.81), p-trend = 0.022. The results were similar for incident CVD; Europeans HR 2.12 (1.14–3.94), p–trend = 0.014; South Asians HR 2.73 (1.20–6.21), p-trend = 0.018. The population attributable fraction in Europeans was 43% for CHD and 28% for CVD. In South Asians it was 63% and 51% respectively.

Conclusions

Lack of adherence to four combined health behaviours was associated with 2 to 3-fold increased risk of incident CVD in Europeans and South Asians. A substantial population impact in the South Asian group indicates important potential for disease prevention in this high-risk group by adherence to healthy behaviours.     

Genetic Variation of COLEC10 and COLEC11 and Association with Serum Levels of Collectin Liver 1 (CL-L1) and Collectin Kidney 1 (CL-K1)

Collectin liver 1 (CL-L1, alias CL-10) and collectin kidney 1 (CL-K1, alias CL-11), encoded by the COLEC10 and COLEC11 genes, respectively, are highly homologous soluble pattern recognition molecules in the lectin pathway of complement. These proteins may be involved in anti-microbial activity and in tissue development as mutations in COLEC11 are one of the causes of the developmental defect syndrome 3MC. We studied variations in COLEC10 and COLEC11, the impact on serum concentration and to what extent CL-L1 and CL-K1 serum concentrations are correlated. We sequenced the promoter regions, exons and exon-intron boundaries of COLEC10 and COLEC11 in samples from Danish Caucasians and measured the corresponding serum levels of CL-L1 and CL-K1. The median concentration of CL-L1 and CL-K1 was 1.87 μg/ml (1.00–4.14 μg/ml) and 0.32 μg/ml (0.11–0.69 μg/ml), respectively. The level of CL-L1 strongly correlated with CL-K1 (ρ = 0.7405, P <0.0001). Both genes were highly conserved with the majority of variations in the non-coding regions. Three non-synonymous variations were tested: COLEC10 Glu78Asp (rs150828850, minor allele frequency (MAF): 0.003), COLEC10 Arg125Trp (rs149331285, MAF: 0.007) and COLEC11 His219Arg (rs7567833, MAF: 0.033). Carriers of COLEC10 Arg125Trp had increased CL-L1 serum levels (P = 0.0478), whereas promoter polymorphism COLEC11-9570C>T (rs3820897) was associated with decreased levels of CL-K1 (P = 0.044). In conclusion, COLEC10 and COLEC11 are highly conserved, which may reflect biological importance of CL-L1 and CL-K1. Moreover, the strong inter individual correlation between the two proteins suggests that a major proportion are found as heterooligomers or subjected to the same regulatory mechanisms.     

Molecular Cloning and Functional Expression of the Equine K+ Channel KV11.1 (Ether à Go-Go-Related/KCNH2 Gene) and the Regulatory Subunit KCNE2 from Equine Myocardium

The KCNH2 and KCNE2 genes encode the cardiac voltage-gated K⁺ channel K_V11.1 and its auxiliary β subunit KCNE2. K_V11.1 is critical for repolarization of the cardiac action potential. In humans, mutations or drug therapy affecting the K_V11.1 channel are associated with prolongation of the QT intervals on the ECG and increased risk of ventricular tachyarrhythmia and sudden cardiac death—conditions known as congenital or acquired Long QT syndrome (LQTS), respectively. In horses, sudden, unexplained deaths are a well-known problem. We sequenced the cDNA of the KCNH2 and KCNE2 genes using RACE and conventional PCR on mRNA purified from equine myocardial tissue. Equine K_V11.1 and KCNE2 cDNA had a high homology to human genes (93 and 88%, respectively). Equine and human K_V11.1 and K_V11.1/KCNE2 were expressed in Xenopus laevis oocytes and investigated by two-electrode voltage-clamp. Equine K_V11.1 currents were larger compared to human K_V11.1, and the voltage dependence of activation was shifted to more negative values with V_1/2 = -14.2±1.1 mV and -17.3±0.7, respectively. The onset of inactivation was slower for equine K_V11.1 compared to the human homolog. These differences in kinetics may account for the larger amplitude of the equine current. Furthermore, the equine K_V11.1 channel was susceptible to pharmacological block with terfenadine. The physiological importance of K_V11.1 was investigated in equine right ventricular wedge preparations. Terfenadine prolonged action potential duration and the effect was most pronounced at slow pacing. In conclusion, these findings indicate that horses could be disposed to both congenital and acquired LQTS.     

Identification and HLA-Tetramer-Validation of Human CD4+ and CD8+ T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4⁺ and CD8⁺ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8⁺ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4⁺ and CD8⁺ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4⁺ and CD8⁺ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4⁺ and 44 CD8⁺ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.     

Burden of Hospital Admission and Repeat Angiography in Angina Pectoris Patients with and without Coronary Artery Disease: A Registry-Based Cohort Study

Aims

To evaluate risk of hospitalization due to cardiovascular disease (CVD) and repeat coronary angiography (CAG) in stable angina pectoris (SAP) with no obstructive coronary artery disease (CAD) versus obstructive CAD, and asymptomatic reference individuals.

Methods and Results

We followed 11,223 patients with no prior CVD having a first-time CAG in 1998–2009 due to SAP symptoms and 5,695 asymptomatic reference individuals from the Copenhagen City Heart Study through registry linkage for 7.8 years (median). In recurrent event survival analysis, patients with SAP had 3–4-fold higher risk of hospitalization for CVD irrespective of CAG findings and cardiovascular comorbidity. Multivariable adjusted hazard ratios(95%CI) for patients with angiographically normal coronary arteries was 3.0(2.5–3.5), for angiographically diffuse non-obstructive CAD 3.9(3.3–4.6) and for 1–3-vessel disease 3.6–4.1(range)(all P<0.001). Mean accumulated hospitalization time was 3.5(3.0–4.0)(days/10 years follow-up) in reference individuals and 4.5(3.8–5.2)/7.0(5.4–8.6)/6.7(5.2–8.1)/6.1(5.2–7.4)/8.6(6.6–10.7) in patients with angiographically normal coronary arteries/angiographically diffuse non-obstructive CAD/1-, 2-, and 3-vessel disease, respectively (all P<0.05, age-adjusted). SAP symptoms predicted repeat CAG with multivariable adjusted hazard ratios for patients with angiographically normal coronary arteries being 2.3(1.9–2.9), for angiographically diffuse non-obstructive CAD 5.5(4.4–6.8) and for obstructive CAD 6.6–9.4(range)(all P<0.001).

Conclusions

Patients with SAP symptoms and angiographically normal coronary arteries or angiographically diffuse non-obstructive CAD suffer from considerably greater CVD burdens in terms of hospitalization for CVD and repeat CAG compared with asymptomatic reference individuals even after adjustment for cardiac risk factors and exclusion of cardiovascular comorbidity as cause. Contrary to common perception, excluding obstructive CAD by CAG in such patients does not ensure a benign cardiovascular prognosis.     

Physical activity attenuates the influence of FTO variants on obesity risk: a meta-analysis of 218,166 adults and 19,268 children

Background

The FTO gene harbors the strongest known susceptibility locus for obesity. While many individual studies have suggested that physical activity (PA) may attenuate the effect of FTO on obesity risk, other studies have not been able to confirm this interaction. To confirm or refute unambiguously whether PA attenuates the association of FTO with obesity risk, we meta-analyzed data from 45 studies of adults (n = 218,166) and nine studies of children and adolescents (n = 19,268).

Methods and Findings

All studies identified to have data on the FTO rs9939609 variant (or any proxy [r ²>0.8]) and PA were invited to participate, regardless of ethnicity or age of the participants. PA was standardized by categorizing it into a dichotomous variable (physically inactive versus active) in each study. Overall, 25% of adults and 13% of children were categorized as inactive. Interaction analyses were performed within each study by including the FTO×PA interaction term in an additive model, adjusting for age and sex. Subsequently, random effects meta-analysis was used to pool the interaction terms. In adults, the minor (A−) allele of rs9939609 increased the odds of obesity by 1.23-fold/allele (95% CI 1.20–1.26), but PA attenuated this effect (p _interaction  = 0.001). More specifically, the minor allele of rs9939609 increased the odds of obesity less in the physically active group (odds ratio  = 1.22/allele, 95% CI 1.19–1.25) than in the inactive group (odds ratio  = 1.30/allele, 95% CI 1.24–1.36). No such interaction was found in children and adolescents.

Conclusions

The association of the FTO risk allele with the odds of obesity is attenuated by 27% in physically active adults, highlighting the importance of PA in particular in those genetically predisposed to obesity. Please see later in the article for the Editors'' Summary